Method of determining the nucleotide sequence of oligonucleotides and DNA molecules

• US 9,212,393 B2
• Filed: 05/03/2011
• Issued: 12/15/2015
• Est. Priority Date: 05/01/1998
• Status: Expired due to Fees

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Williams, Peter
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/099,718
• Publication Number:
  US 20110294115A1
• Time in Patent Office: 1,687 Days
• Field of Search: 435/6.1, 435/91.1, 435/91.2, 422/68.1, 422/82.01, 536/24.3
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2563/103 luminescence
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes
• CPC Class Codes: G01N 33/6863 Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors

Abstract

The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.

Claims

1. An apparatus for DNA sequencing comprising:

(a) at least one reaction chamber including a DNA primer/template system which produces a detectable signal when a DNA polymerase enzyme incorporates an unlabeled and unblocked deoxyribonucleotide monophosphate onto the 3′

end of the primer strand;

(b) a system for introducing into, and evacuating from, said reaction chamber at least one reagent selected from the group consisting of;

buffers, electrolytes, DNA template, DNA primer, deoxyribonucleotides, and polymerase enzymes;

(c) a system for converting said detectable signal into an electrical signal based on an electrical potential generated from said detectable signal in the at least one reaction chamber; and

(d) a system for measuring the electrical signal generated by the detectable signal produced in the at least one reaction chamber.

2. The apparatus of claim 1, wherein said at least one reaction chamber further comprises said deoxyribonucleotide monophosphate, and wherein said deoxyribonucleotide monophosphate is not chemically modified.

3. The apparatus of claim 1, further comprising (e) a voltmeter configured to measure said electrical signal.

4. The apparatus of claim 1, wherein said at least one chamber further includes a solid support, wherein said primer/template system comprises a primer and a template, and wherein said template is tethered to said solid support.

5. The apparatus of claim 4, wherein said template comprises a linker moiety for tethering to said solid support.

6. The apparatus of claim 1, wherein said DNA polymerase lacks 5′

to 3′

exonuclease activity.

7. The apparatus of claim 1, wherein the amplitude of said electrical signal corresponds to the number of nucleotides added to said primer strand by said DNA polymerase.

8. The apparatus of claim 1, wherein said apparatus provides real-time detection of incorporation of said deoxyribonucleotide monophosphate onto said primer strand.

9. A method of DNA sequencing comprising:

(a) providing a primer/template system comprising a template sequence hybridized to a primer oligonucleotide in the presence of a DNA polymerase;

(b) contacting said primer/template system with a single type of deoxyribonucleotide under conditions that produce a detectable signal when said DNA polymerase incorporates an unlabeled and unblocked deoxyribonucleotide onto the 3′

end of said primer oligonucleotide, wherein the contacting occurs in a reaction chamber;

(c) converting, with a device, said detectable signal into an electrical signal based on an electrical potential generated across said device by said detectable signal, wherein the converting occurs in the reaction chamber; and

(d) detecting the electrical signal generated by the detectable signal produced in the reaction chamber.

10. The method of claim 9, further comprising (e) measuring said electrical signal with a voltmeter.

11. The method of claim 9, further comprising (e) flushing said reaction chamber with dNTP free buffer.

12. The method of claim 11, further comprising repeating steps (b) through (e).

13. The method of claim 11, further comprising repeating steps (b) through (e) until the complete nucleotide sequence of said template sequence is determined.

14. The method of claim 9, wherein said deoxyribonucleotide is not chemically modified.

15. The method of claim 9, wherein said reaction chamber further includes a solid support, and wherein said template sequence is tethered to said solid support.

16. The method of claim 15, wherein said template sequence comprises a linker moiety for tethering to said solid support.

17. The method of claim 9, wherein said DNA polymerase lacks 5′

to 3′

exonuclease activity.

18. The method of claim 9, wherein the amplitude of said electrical signal corresponds to the number of nucleotides added to said primer strand by said DNA polymerase.

19. The method of claim 9, wherein said electrical signal provides real-time detection of incorporation of said deoxyribonucleotide monophosphate onto said primer strand by said DNA polymerase.